Methods for producing retinal tissue and retina-related cell

ABSTRACT

The invention provides a method for producing a retinal tissue by (1) subjecting pluripotent stem cells to floating culture in a serum-free medium containing a substance inhibiting the Wnt signal pathway to form an aggregate of pluripotent stem cells, (2) subjecting the aggregate to floating culture in a serum-free medium containing a basement membrane preparation, and then (3) subjecting the aggregate to floating culture in a serum-containing medium. The invention also provides a method for producing an optic-cup-like structure, a method for producing a retinal pigment epithelium, and a method for producing a retinal layer-specific neural cell.

TECHNICAL FIELD

The present invention relates to a method for producing a retinaltissue, and retina-related cells such as retinal layer-specific nervecell, retinal pigment epithelium, and so on.

BACKGROUND ART

The central nervous system tissues such as the brain and retina have lowregenerative capacity and damaged tissues scarcely recoverspontaneously. Therefore, regenerative medicine that transplants cellsdifferentiated from pluripotent stem cells for the treatment is expectedto be the last card for overcoming intractable diseases. Furthermore,human-derived cells obtained by differentiation from pluripotent stemcells are considered to be able to accurately evaluate the effects ofchemical substances on human, and the research and development towardapplication to toxicity evaluation of compounds and drug discovery areunderway.

Retina is an important sensory tissue that receives light, converts itto electrical signals and, after information processing, conveys theinformation via axons to the visual center of the brain. Retina islargely made of two inside and outside epithelial tissues superimposedon top of each other. The inside is a neural retina that receives lightand processes information, and contains more than one type of cell suchas photoreceptor. The outside is a retinal pigment epithelium which is amonolayer cell sheet that supports the survival and function of thephotoreceptors.

There have been known reports on the production of retinallayer-specific nerve cells constituting the neural retina(photoreceptors, horizontal cells, amacrine cells, ganglion cells and soon) from pluripotent stem cells (patent document 1). Furthermore, as amethod for producing a three-dimensional retinal tissue from pluripotentstem cells, it is described that retinal progenitor tissue,optic-cup-like structure and multi-layer neural retinal tissue can beproduced in vitro by forming homogeneous pluripotent stem cellaggregates in a serum-free medium and subjecting them to floatingculture in the presence of a basement membrane preparation (non-patentdocument 1 and patent document 2).

On the other hand, a report on the production of retinal pigmentepithelia by using pluripotent stem cells is also known (non-patentdocument 2). However, there is no report on the production of retinalpigment epithelia with high efficiency.

DOCUMENT LIST Patent Documents

-   patent document 1: WO 2008/087917-   patent document 2: WO 2011/055855

Non-Patent Documents

-   non-patent document 1: Mototsugu Eiraku, Nozomu Takata, Hiroki    Ishibashi, Masako Kawada, Eriko Sakakura, Satoru Okuda, Kiyotoshi    Sekiguchi, Taiji Adachi & Yoshiki Sasai (2011) Self-organizing    optic-cup morphogenesis in three-dimensional culture. Nature Volume:    472, Pages: 51-56-   non-patent document 2: Maria Idelson, Ruslana Alper, Alexey    Obolensky, Etti Ben-Shushan, Itzhak Hemo, Nurit Yachimovich-Cohen,    Hanita Khaner, Yoav Smith, Ofer Wiser, Michal Gropp, Malkiel A.    Cohen, Sharona Even-Ram, Yael Berman-Zaken, Limor Matzrafi, Gideon    Rechavi, Eyal Banin, and Benjamin Reubinoff (2009) Directed    Differentiation of HumanEmbryonic Stem Cells into Functional Retinal    Pigment Epithelium Cells. Cell Stem Cell 5, 396-408

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

A method for producing retinal tissue, optic-cup-like structure andretinal layer-specific nerve cell, and retinal pigment epithelium withhigher efficiency has been demanded.

Means of Solving the Problems

The present inventors have conducted intensive studies in view of suchsituation and arrived at the present invention.

Accordingly, the present invention is as follows.

[1] A method for producing a retinal tissue, comprising the followingsteps (1) to (3):(1) a first step of subjecting pluripotent stem cells to floatingculture in a serum-free medium containing a substance inhibiting the Wntsignal pathway to form an aggregate of pluripotent stem cells,(2) a second step of subjecting the aggregate formed in the first stepto floating culture in a serum-free medium containing a basementmembrane preparation, and(3) a third step of subjecting the aggregate cultured in the second stepto floating culture in a serum-containing medium.[2] A method for producing an optic-cup-like structure, comprising astep of subjecting the retinal tissue obtained by the method of theaforementioned [1] to floating culture in a serum-free medium orserum-containing medium each containing a substance acting on the Sonichedgehog (hereinafter, sometimes referred to as “Shh”) signal pathwayand a substance acting on the Wnt signal pathway.[3] A method for producing a retinal pigment epithelium, comprising astep of subjecting the retinal tissue obtained by the method of theaforementioned [1] to floating culture in a serum-free medium orserum-containing medium each containing a substance acting on the Wntsignal pathway (wherein the aforementioned serum-free medium andserum-containing medium are free of a substance acting on the Sonichedgehog (hereinafter, sometimes referred to as “Shh”) signal pathway).[4] The method of the aforementioned [3], wherein the aforementionedserum-free medium or serum-containing medium containing a substanceacting on the Wnt signal pathway further comprises a substance acting onthe Activin signal pathway.[5] The method of any of the aforementioned [1] to [4], wherein theaforementioned pluripotent stem cells are primate pluripotent stemcells.[6] The method of any of the aforementioned [1] to [4], wherein theaforementioned pluripotent stem cells are human pluripotent stem cells.[7] The method of any of the aforementioned [1] to [6], wherein theaforementioned basement membrane preparation is at least oneextracellular matrix molecule selected from the group consisting oflaminin, type IV collagen, heparan sulfate proteoglycan and entactin.[8] The method of any of the aforementioned [1] to [7], wherein theaforementioned first step to the third step are performed in thepresence of Knockout serum replacement (hereinafter, sometimes referredto as “KSR”).[9] A method for producing a retinal layer-specific neural cell,comprising bringing a retinal progenitor cell contained in a retinaltissue derived from a primate pluripotent stem cell into contact with asubstance inhibiting the Notch signal pathway.[10] The method of the aforementioned [9], wherein the aforementionedsubstance inhibiting the Notch signal pathway is a gamma secretaseactivity inhibitory substance.[11] The method of the aforementioned [9] or [10], wherein theaforementioned substance inhibiting the Notch signal pathway isN—[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester(hereinafter, sometimes referred to as “DAPT”).[12] The method of any of the aforementioned [9] to [11], wherein theaforementioned primate is human.[13] The method of any of the aforementioned [9] to [12], wherein theretinal layer-specific neural cell is a photoreceptor.[14] The method of any of the aforementioned [9] to [12], wherein theretinal layer-specific neural cell is a ganglion cell.[15] The method of any of the aforementioned [9] to [14], wherein theretinal progenitor cell contained in the retinal tissue derived from aprimate pluripotent stem cell is a retinal progenitor cell contained ina retinal tissue produced by the following steps:(1) a first step of subjecting primate pluripotent stem cells tofloating culture in a serum-free medium containing a substanceinhibiting the Wnt signal pathway to form an aggregate of primatepluripotent stem cells,(2) a second step of subjecting the aggregate formed in the first stepto floating culture in a serum-free medium containing a basementmembrane preparation,(3) a third step of subjecting the aggregate cultured in the second stepto floating culture in a serum-containing medium,(4) a fourth step of subjecting the aggregate cultured in the third stepto floating culture in a serum-free medium or serum-containing mediumeach containing a substance acting on the Sonic hedgehog (hereinafter,sometimes referred to as “Shh”) signal pathway and a substance acting onthe Wnt signal pathway, to form an optic-cup-like structure, and(5) a step of subjecting the optic-cup-like structure formed in thefourth step to floating culture.[16] A reagent for evaluating toxicity or drug efficacy, comprising aretinal tissue, an optic-cup-like structure, a retinal pigmentepithelium or a retinal layer-specific neural cell produced by themethod of any of the aforementioned [1] to [15].[17] A method for evaluating toxicity or drug efficacy of a testsubstance, comprising bringing a retinal tissue, an optic-cup-likestructure, a retinal pigment epithelium or a retinal layer-specificneural cell produced by the method of any of the aforementioned [1] to[15] into contact with the test substance, and examining the influenceof the substance on the tissue, structure or cell.[18] A therapeutic agent for a disease due to a disorder of a retinaltissue, comprising a retinal tissue, an optic-cup-like structure, aretinal pigment epithelium or a retinal layer-specific neural cellproduced by the method of any of the aforementioned [1] to [15].[19] A method for treating a disease due to a disorder of a retinaltissue, comprising transplanting an effective amount of a retinaltissue, an optic-cup-like structure, a retinal pigment epithelium or aretinal layer-specific neural cell produced by the method of any of theaforementioned [1] to [15] to a target in need of the transplantation.[20] A retinal tissue, an optic-cup-like structure, a retinal pigmentepithelium or a retinal layer-specific nerve cell produced by the methodof any of the aforementioned [1] to [15] for use in the treatment of adisease due to a disorder of a retinal tissue.

Effect of the Invention

According to the present invention, a retinal tissue, optic-cup-likestructure, retinal layer-specific nerve cell or retinal pigmentepithelium can be produced at high efficiency. Accordingly, in view ofefficient provision of a retinal tissue, optic-cup-like structure,retinal layer-specific nerve cell or retinal pigment epithelium for thepurpose of toxicity or drug efficacy evaluation of a chemical substance,etc., a transplantation treatment and so on, the present invention ishighly useful.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view that shows a bright-field image (A) and a fluorescenceimage (B) of human pluripotent stem cell-derived aggregates, which wereproduced by adding Matrigel (hereinafter, sometimes referred to asMatrigel) alone and without adding a substance inhibiting the Wnt signalpathway, on day 25 from the start of the floating culture, abright-field image (C) and a fluorescence image (D) of human pluripotentstem cell-derived aggregates, which were produced by adding a substanceinhibiting the Nodal signal pathway and Matrigel, on day 25 from thestart of the floating culture, and a bright-field image (E) and afluorescence image (F) of human pluripotent stem cell-derivedaggregates, which were produced by adding a substance inhibiting the Wntsignal pathway and Matrigel, on day 25 from the start of the floatingculture.

FIG. 2 is a view that shows a bright-field image (A) and a fluorescenceimage (B) of aggregates on day 18 from the start of the floatingculture, which were produced by floating culture of human pluripotentstem cells by adding a substance inhibiting the Wnt signal pathway,floating culture thereof in the presence of Matrigel and furtherfloating culture thereof in the absence of fetal calf serum, abright-field image (C) and a fluorescence image (D) of aggregates on day18 from the start of the floating culture, which were produced byfloating culture of human pluripotent stem cells by adding a substanceinhibiting the Wnt signal pathway, floating culture thereof in thepresence of Matrigel and further floating culture thereof in thepresence of fetal calf serum, and a bright-field image (E) and afluorescence image (F) of aggregates on day 18 from the start of thefloating culture, which were produced by floating culture of humanpluripotent stem cells by adding a substance inhibiting the Wnt signalpathway, floating culture thereof in the presence of Matrigel andfurther floating culture thereof in the presence of fetal calf serum anda substance acting on the Sonic hedgehog (hereinafter, sometimesreferred to as “Shh”) signal pathway.

FIG. 3 shows FACS histograms (A) of GFP-expressing cell constitutingaggregates on day 18 from the start of the floating culture, which wereproduced by floating culture of human pluripotent stem cells by adding asubstance inhibiting the Wnt signal pathway, floating culture thereof inthe presence of Matrigel and further floating culture thereof in theabsence of fetal calf serum, FACS histograms (B) of GFP-expressing cellconstituting aggregates on day 18 from the start of the floatingculture, which were produced by floating culture of human pluripotentstem cells by adding a substance inhibiting the Wnt signal pathway,floating culture thereof in the presence of Matrigel and furtherfloating culture thereof in the presence of fetal calf serum, FACShistograms (C) of GFP-expressing cell constituting aggregates on day 18from the start of the floating culture, which were produced by floatingculture of human pluripotent stem cells by adding a substance inhibitingthe Wnt signal pathway, floating culture thereof in the presence ofMatrigel and further floating culture thereof in the presence of fetalcalf serum and a substance inhibiting the Shh signal pathway, and agraph (D) showing the proportion of GFP strong positive cells, which areretinal progenitor cells.

FIG. 4 is a view that shows the results of immunostaining of retinaltissues (A, B, C) on day 60 from the start of the floating culture andretinal tissues (D, E, F, G, H, I) on day 126 from the start of thefloating culture, which were produced by the production method ofretinal tissue of the present invention.

FIG. 5 is a view that shows images of an overlap of a bright-field and afluorescence image of the same part of an aggregate on day 14 (A), day15 (B), day 16 (C), day 17 (D) from the start of the floating culture bythe production method of an optic-cup-like structure of the presentinvention.

FIG. 6 is a view that shows a bright-field image (A) and a fluorescenceimage (B) by microscopic observation, and a two-photon microscopeobservation image (C) and a 3D reconstitution image (D) obtained from atwo-photon microscopic observation image, of a floating culturedaggregate on days 24 to 26 from the start of the floating culture by theproduction method of an optic-cup-like structure of the presentinvention.

FIG. 7 is a view that shows the results of immunostaining of a frozensection of an optic-cup-like structure produced by floating culture bythe production method of an optic-cup-like structure of the presentinvention, with an anti-Mitf antibody and an anti-Chx10 antibody.

FIG. 8 is a view that shows a fluorescence microscope (A) in the case offloating culture until day 41 from the start of the floating culture,the start of the floating culture under the conditions without additionof 10 μM DAPT, a fluorescence microscope (B) in the case of floatingculture until day 41 from the start of the floating culture under theconditions with addition of DAPT, photograph of a frozen sectionimmunostained with an anti-Recoverin antibody (C) or with an anti-Brn3antibody (E) in the case of floating culture until day 43 from the startof the floating culture under the conditions without addition of 10 μMDAPT, and photograph of frozen section immunostained with ananti-Recoverin antibody (D) or with an anti-Brn3 antibody (F) in thecase of floating culture until day 43 of differentiation induction underthe conditions with addition of DAPT, each of which using a Crx::GFPknock-in human ES cell-derived retinal tissue on day 29 from the startof the floating culture.

FIG. 9 is a view that shows a fluorescence microscope (A) in the case offloating culture, until day 49 from the start of the floating cultureunder the conditions without addition of 10 μM DAPT, a fluorescencemicroscope (B) in the case of floating culture until day 49 from thestart of the floating culture under the conditions with addition ofDAPT, photograph of a frozen section immunostained with ananti-Recoverin antibody (C) or with an anti-Brn3 antibody (E) in thecase of floating culture until day 49 from the start of the floatingculture under the conditions without addition of 10 μM DAPT, andphotograph of frozen section immunostained with an anti-Recoverinantibody (D) or with an anti-Brn3 antibody (F) in the case of floatingculture until day 49 from the start of the floating culture under theconditions with addition of DAPT, of a Crx::GFP knock-in human EScell-derived retinal tissue on day 38 from the start of the floatingculture after freeze-thawing the retinal tissue on day 33 from the startof the floating culture.

FIG. 10 is a view that shows a bright-field of an aggregate produced byfloating culture in a serum-free medium containing a substanceinhibiting the Wnt signal pathway to form an aggregate, floating cultureof the formed aggregate in a serum-free medium in the presence of abasement membrane preparation, culturing the cultured aggregate in amedium containing a serum, and culturing the cultured aggregate in amedium containing a substance acting on the Wnt signal pathway.

FIG. 11 is a view that shows a bright-field of an aggregate produced byfloating culture in a serum-free medium containing a substanceinhibiting the Wnt signal pathway to form an aggregate, floating cultureof the formed aggregate in a serum-free medium in the presence of abasement membrane preparation, culturing the cultured aggregate in amedium containing a serum, and culturing the cultured aggregate in amedium containing a substance acting on the Wnt signal pathway and asubstance acting on the Activin A pathway.

MODE(S) FOR CARRYING OUT THE INVENTION

Mode(s) for carrying out the present invention is explained in detailbelow.

In the present invention, the “transformant” means the entirety or apart of the living matter such as cell produced by transformation.Examples of the transformant include prokaryotic cell, yeast, animalcell, plant cell, insect cell and so on. Depending on the target, thetransformant is also sometimes called transformed cell, transformedtissue, transformed host and so on. The cell used in the presentinvention may also be a transformant.

Examples of the prokaryotic cell used for genetically-engineeredtechnique in the present invention include prokaryotic cells belongingto the genus Escherichia, the genus Serratia, the genus Bacillus, thegenus Brevibacterium, the genus Corynebacterium, the genusMicrobacterium, the genus Pseudomonas and so on, such as EscherichiaXL1-Blue, Escherichia XL2-Blue, and Escherichia DH1. These cells arespecifically described in, for example, “Molecular Cloning (3rdedition)” by Sambrook, J and Russell, D. W., Appendix 3 (Volume 3),Vectors and Bacterial strains. A3.2 (Cold Spring Harbor USA 2001).

The “vector” in the present invention means a vector capable oftransferring a desired polynucleotide sequence into an object cell.Examples of such vector include those capable of autonomouslyreplicating in a host cell such as prokaryotic cell, yeast, animal cell,plant cell, insect cell, animal individual and plant individual, orcapable of being incorporated into a chromosome, and containing apromoter at a position suitable for polynucleotide transcription.

Of such vectors, a vector suitable for cloning is sometimes indicated asa “cloning vector”. Such cloning vector generally has multiple cloningsites containing a plurality of restriction enzyme sites. At present,there are many vectors usable for gene cloning in the pertinent field,and they are sold by distributors with different names since they areslightly different (e.g., kind and sequence of restriction enzymes atmulti cloning sites). For example, representative ones are described(distributors are also described) in “Molecular Cloning (3rd edition)”by Sambrook, J and Russell, D. W., Appendix 3 (Volume 3), Vectors andBacterial strains. A3.2 (Cold Spring Harbor USA, 2001), and those ofordinary skill in the art can use them as appropriate according to theobject.

The “vector” in the present invention also includes “expression vector”,“reporter vector”, and “recombinant vector”. The “expression vector”means a nucleic acid sequence wherein various regulatory elements inaddition to a structural gene and a promoter that regulates theexpression thereof are linked in such a manner that they can be operablein the host cell. Examples of the “regulatory element” includeterminator, selection marker such as a drug resistance gene, and onecontaining an enhancer. It is well known to those of ordinary skill inthe art that the type of an expression vector of living matter (e.g.,animal) and the kind of the regulatory element to be used may varydepending on the host cell.

Examples of the “recombinant vector” in the present invention include(a) lambda FIX vector (phage vector) for screening for genomic library,(b) lambda ZAP vector (phage vector) for screening for cDNA, and (c)pBluescript II SK+/−, pGEM, and pCR2.1 vector (plasmid vector) forcloning of genomic DNA. Examples of the “expression vector” includepSV2/neo vector, pcDNA vector, pUC18 vector, pUC19 vector, pRc/RSVvector, pLenti6/V5-Dest vector, pAd/CMV/V5-DEST vector, pDON-AI-2/neovector, and pMEI-5/neo vector (plasmid vector) and so on. Examples ofthe “reporter vector” include pGL2 vector, pGL3 vector, pGL4.10 vector,pGL4.11 vector, pGL4.12 vector, pGL4.70 vector, pGL4.71 vector, pGL4.72vector, pSLG vector, pSLO vector, pSLR vector, pEGFP vector, pAcGFPvector, pDsRed vector and so on. These vectors can be utilized asappropriate by reference to the aforementioned Molecular Cloningreference.

As a technique for introducing a nucleic acid molecule into a cell inthe present invention, for example, transformation, transduction,transfection and so on can be mentioned. As such introduction technique,for example, the methods described in Ausubel F. A. et al. ed. (1988),Current Protocols in Molecular Biology, Wiley, New York, N.Y.; SambrookJ. et al. (1987), Molecular Cloning: A Laboratory Manual, 2nd Ed. and3rd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;extra issue, Experimental Medicine “transgene & expression analysisexperiment method” YODOSHA CO., LTD., 1997, and so on can bespecifically mentioned. As the technique for confirming intracellularintroduction of a gene, for example, Northern blot analysis, Westernblot analysis and other well-known conventional techniques and so on canbe mentioned.

In the present invention, the “stem cell” refers to a cell thatmaintains the same differentiation capacity even after cell division,and a tissue thereof can be regenerate when the tissue is injured. Here,the stem cell may be an embryonic stem cell (ES cell) or a tissue stemcell (also called tissular stem cell, tissue-specific stem cell orsomatic stem cell), or an artificial pluripotent stem cell (iPS cell:induced pluripotent stem cell) but is not limited thereto. As isappreciated from the fact that the above-mentioned stem cell-derivedtissue cell can regenerate a tissue, it is known that the stem cell candifferentiate into a normal cell close to one in a living body.

Stem cells are available from given organizations, or a commerciallyavailable product can also be purchased. For example, human embryonicstem cells, KhES-1, KhES-2 and KhES-3, are available from KyotoUniversity's Institute for Frontier Medical Sciences. EB5 cell, which isa mouse embryonic stem cell, is available from RIKEN, and D3 cell lineis available from ATCC.

Stem cells can be maintained by culturing according to a method knownper se. For example, stem cells can be maintained by feeder cell-freeculture supplemented with fetal calf serum (FCS), Knockout SerumReplacement (KSR), and LIF.

In the present invention, the “pluripotent stem cell” refers to a stemcell that can be cultured in vitro and has an ability to differentiateinto any cell (triploblast (ectoderm, mesoderm, endoderm)-derivedtissue) constituting a living body except for placenta (differentiationpluripotency), including an embryonic stem cell (ES cell). The“pluripotent stem cell” is obtained from fertilized egg, clone embryo,reproductive stem cell, and stem cell in a tissue. It also includes acell having artificial differentiation pluripotency similar to that ofembryonic stem cells, after introducing several kinds of genes into asomatic cell (also called artificial pluripotent stem cell). Pluripotentstem cell can be produced by a method known per se. Examples of theproduction method include the methods described in Cell 131(5) pp.861-872, Cell 126(4) pp. 663-676 and so on.

In the present invention, the “embryonic stem cell (ES cell)” refers toa stem cell having a self replication ability and multipotency (i.e.,“pluripotency”), which is a pluripotent stem cell derived from an earlyembryo. Embryonic stem cell was first established in 1981, and has alsobeen applied to the generation of knockout mouse since 1989. In 1998, ahuman embryonic stem cell was established, which is also being utilizedfor regenerative medicine.

In the present invention, the “artificial pluripotent stem cell” refersto a cell induced to have multipotency by directly reprogramming adifferentiated cell such as fibroblast etc. by the expression of severalkinds of genes such as Oct3/4, Sox2, Klf4, and Myc, which wasestablished by Yamanaka et al. in mouse cell in 2006 (Takahashi K,Yamanaka S. Cell. 2006, 126(4), p 663-676). In 2007, it was alsoestablished in human fibroblast, and has multipotency similar to that ofembryonic stem cells (Takahashi K, Tanabe K, Ohnuki M, Narita M,Ichisaka T, Tomoda K, Yamanaka S. Cell. 2007, 131(5), p 861-872.; Yu J,Vodyanik M A, Smuga-Otto K, Antosiewicz-Bourget J, Frane J L, Tian S,Nie J, Jonsdottir G A, Ruotti V, Stewart R, Slukvin I I, Thomson J A.,Science. 2007, 318(5858), p 1917-1920.; Nakagawa M, Koyanagi M, TanabeK, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N,Yamanaka S. Nat Biotechnol., 2008, 26(1), p 101-106).

A genetically-modified pluripotent stem cell can be produced, forexample, using a homologous recombination technique. Examples of thegene on the chromosome, which is to be modified for the production of amodified pluripotent stem cell, include a histocompatibility antigengene, a gene related to a disease due to a disorder of nerve system celland so on. A target gene on the chromosome can be modified by themethods described in Manipulating the Mouse Embryo, A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press (1994); GeneTargeting, A Practical Approach, IRL Press at Oxford University Press(1993); Bio Manual series 8, gene targeting, Production of mutant mouseby using ES cells, YODOSHA CO., LTD. (1995) and so on.

To be specific, for example, the genomic gene of a target gene to bemodified (e.g., histocompatibility antigen gene, disease-related geneand so on) is isolated, and a target vector used for homologousrecombination of the target gene is produced using the isolated genomicgene. The produced target vector is introduced into stem cells, andcells showing homologous recombination between the target gene and thetarget vector are selected, whereby stem cells having modified gene onthe chromosome can be produced.

As a method for isolating the genomic gene of the target gene, knownmethods described in Molecular Cloning, A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press (1989), Current Protocolsin Molecular Biology, John Wiley & Sons (1987-1997) and so on can bementioned. Moreover, the genomic gene of the target gene can be isolatedusing genomic DNA library screening system (manufactured by GenomeSystems), Universal GenomeWalker Kits (manufactured by CLONTECH) and soon.

A target vector used for homologous recombination of the target gene canbe produced, and a homologous recombinant can be efficiently selectedaccording to the methods described in Gene Targeting, A PracticalApproach, IRL Press at Oxford University Press (1993); Bio Manual series8, gene targeting, Production of mutant mouse by using ES cells, YODOSHACO., LTD. (1995) and so on. The target vector may be any of replacementtype and insertion type, and the selection method may be positiveselection, promoter selection, negative selection, polyA selection andso on.

As a method for selecting an object homologous recombinant from theselected cell lines, Southern hybridization method, PCR method and so onfor genomic DNA can be mentioned.

In the present invention, the “tissue” refers to a structure of a cellpopulation, which has a conformation wherein more than one type of celldifferent in the shape and property are sterically configured in a givenpattern.

In the present invention, the “retinal tissue” means a retinal tissuewherein at least two or more types of cells such as photoreceptors,horizontal cells, bipolar cells, amacrin cells, retinal ganglion cells,their precursor cells or retinal progenitor cells thereof, whichconstitute respective retinal layers in living retina, are stericallyarranged in layers. With regard to each cell, which cell constituteswhich retinal layer can be confirmed by a known method, for example, theexpression of a cell marker.

Examples of the retina cell marker include, but are not limited to, Rax(progenitor cell of retina), PAX6 (progenitor cell), nestin (expressedin progenitor cell of hypothalamus neuron but not expressed in retinalprogenitor cell), Sox1 (expressed in hypothalamus neuroepithelium butnot expressed in retina), Crx (precursor cell of photoreceptor), and soon. In particular, examples of the marker of the above-mentioned retinallayer-specific neuron include, but are not limited to, Chx10 (bipolarcell), L7 (bipolar cell), Tuj1 (ganglion cell), Brn3 (ganglion cell),Calretinin (amacrine cell), Calbindin (horizontal cell), Rhodopsin(photoreceptor), Recoverin (photoreceptor), RPE65 (pigment epithelium),Mitf (pigment epithelium) Nrl (rod cell), Rxr-gamma (cone cell) and soon.

In the present invention, the “optic-cup-like structure” refers to astructure having a shape similar to that of the optic cup in thedevelopment process of embryo. In the development process of embryo, theprimordium of retina is developed from the side face of diencephalon,and formed like a pouch protruding from the diencephalon. The pouch-likeepithelial structure is called an optic vesicle. The outermost part ofthe optic vesicle (to be the neural retina in the future) graduallyinvaginates toward the inside of the optic vesicle, and forms an opticcup which is a cup-like tissue composed of two inside and outside layersof epithelium. The optic cup thereafter grows large and forms the retinawith a retinal tissue. Whether it is an optic-cup-like structure can beconfirmed by those of ordinary skill in the art through observation witha microscope, a magnifying glass and so on.

The optic-cup-like structure to be produced in the present inventionshows not only a morphologically optic cup-like protrusion but alsohighly frequent expression of Rax, which is a retinal progenitor cellmarker, in the cells constituting the optic-cup-like structure. Inaddition, the outer layer of the optic-cup-like structure also shows alayer of retinal pigment epithelia expressing Mitf, and the inner layershows cells constituting retinal tissues, such as retinal progenitorcells expressing Chx10. Such optic-cup-like structure closely resemblesthe structure of optic cup tissues in the development of a living body.

The “retinal layer” in the present invention means each layerconstituting the retina. Specific examples thereof include retinalpigment epithelial layer, photoreceptor layer, external limitingmembrane, outer nuclear layer, outer plexiform layer, inner nuclearlayer, inner plexiform layer, ganglion cell layer, nerve fiber layer andinner limiting membrane.

The “retinal layer-specific neural cell” in the present invention meansa neural cell constituting a retinal layer and specific to the retinallayer.

The “retinal progenitor cell” in the present invention refers to aprogenitor cell that can be differentiated into any mature retinal cellof a photoreceptor, a horizontal cell, a bipolar cell, an amacrine cell,and a retinal ganglion cell.

On the other hand, the photoreceptor precursor, horizontal precursorcell, bipolar precursor cell, amacrine precursor cell, and retinalganglion precursor cell are precursor cells determined to differentiateinto a photoreceptor, a horizontal cell, a bipolar cell, an amacrinecell, and a retinal ganglion cell, respectively.

The “retinal pigment epithelium” in the present invention means anepithelial cell present on the outer side of the neural retinal tissuein the retina of a living body. Whether or not a retinal pigmentepithelium can be easily confirmed by those of ordinary skill in the artby, for example, expression of a cell marker (RPE65 (pigmentepithelium), Mitf (pigment epithelium), etc.), the presence of melaningranule, characteristic polygonal cell form and so on.

The medium to be used in the present invention can be prepared from amedium used for culture of animal cell as a basal medium. Examples ofthe basal medium include BME medium, BGJb medium, CMRL1066 medium,Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium,Medium199 medium, Eagle MEM medium, αMEM medium, DMEM medium, hammedium, RPMI1640 medium, Fischer's medium, and mixed medium thereof, andthe medium is not particularly limited as long as it can be used forculturing animal cells.

The “serum-free medium” in the present invention means a medium free ofunadjusted or unpurified serum. A medium containing purifiedblood-derived components and animal tissue-derived components (e.g.,growth factor) is considered a serum-free medium unless unadjusted orunpurified serum is contained therein.

The medium is not particularly limited as long as it is as definedabove. However, to avoid complicated preparation, a serum-free medium(GMEM or DMEM, 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acidMix, 1 mM sodium pyruvate) added with an appropriate amount (e.g.,1-20%) of commercially available KSR can be used as the serum-freemedium.

In addition, the serum-free medium may contain a serum replacement. Theserum replacement can appropriately contain, for example, albumin,transferrin, fatty acid, collagen precursor, trace element,2-mercaptoethanol or 3′ thiolglycerol, an equivalent thereof and so on.Such serum replacement can be prepared by, for example, the methoddescribed in WO98/30679. In addition, to perform the method of thepresent invention more conveniently, the serum replacement can be acommercially available product. Examples of such commercially availableserum replacement include Chemically-defined Lipid concentrated(manufactured by Gibco), and Glutamax (manufactured by Gibco).

The serum-free medium to be used for floating culture can contain fattyacid or lipid, amino acid (e.g., non-essential amino acid), vitamin,growth factor, cytokine, antioxidant, 2-mercaptoethanol, pyruvic acid,buffering agent, inorganic salts and so on.

The “serum-containing medium” in the present invention means a mediumcontaining unadjusted or unpurified serum. The medium is notparticularly limited as long as it is as defined above. In addition, theserum-containing medium can contain fatty acid or lipid, amino acid(e.g., non-essential amino acid), vitamin, growth factor, cytokine,antioxidant, 2-mercaptoethanol, pyruvic acid, buffering agent, inorganicsalts and so on.

The “floating culture” in the present invention means cultivating underconditions prohibiting adhesion of cell or cell mass to a cell culturevessel material and so on.

The cell culture vessel to be used in floating culture is notparticularly limited as long as it enables “floating culture”, and thoseof ordinary skill in the art can appropriately determine same. Examplesof such cell culture vessel include flask, tissue culture flask, dish,petri dish, tissue culture dish, multidish, microplate, microwell plate,micropore, multiplate, multiwell plate, chamber slide, schale, tube,tray, culture bag, and roller bottle. Since these cell culture vesselsare used for floating culture, they are preferably cell non-adhesive. Asa cell non-adhesive vessel, one having its surface not artificiallytreated to improve cell adhesiveness (e.g., coating treatment withextracellular matrix, etc.) and so on can be used.

The “primates” in the present invention mean mammals belonging toprimate. Examples of the primates include Strepsirrhini such as lemur,loris, and Tsubai, and Haplorhini such as monkey, anthropoid ape, andhuman.

<Production Method of Retinal Tissue>

The first aspect of the present invention is a method for producing aretinal tissue, comprising the following steps (1) to (3):

(1) a first step of subjecting pluripotent stem cells to floatingculture in a serum-free medium containing a substance inhibiting the Wntsignal pathway to form an aggregate of pluripotent stem cells,(2) a second step of subjecting the aggregate formed in the first stepto floating culture in a serum-free medium containing a basementmembrane preparation, and(3) a third step of subjecting the aggregate cultured in the second stepto floating culture in a serum-containing medium.

(1) First Step

The first step of subjecting pluripotent stem cells to floating culturein a serum-free medium containing a substance inhibiting the Wnt signalpathway to form an aggregate of pluripotent stem cells is explained.

A substance inhibiting the Wnt signal pathway is not particularlylimited as long as it can suppress signal transduction mediated by Wnt.Examples of the substance inhibiting the Wnt signal pathway includeDkk1, Cerberus protein, Wnt receptor inhibitor, soluble-type Wntreceptor, Wnt antibody, casein kinase inhibitor, dominant negative Wntprotein, CKI-7 (N-(2-aminoethyl)-5-chloro-isoquinoline-8-sulfonamide),D4476(4-{4-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl}benzamide),IWR-1-endo (IWR1e), IWP-2 and so on.

The concentration of the substance inhibiting the Wnt signal pathway tobe used in the present invention only needs to be a concentration atwhich aggregates of pluripotent stem cells are formed. For example, acommon substance inhibiting the Wnt signal pathway such as IWR1e isadded at a concentration of about 0.1 μM to 100 μM, preferably about 1μM to 10 μM, more preferably about 3 μM.

A substance inhibiting the Wnt signal pathway may be added to serum-freemedium before the start of the floating culture, or added to aserum-free medium within several days from the start of the floatingculture (e.g., within 5 days). Preferably, a substance inhibiting theWnt signal pathway is added to a serum-free medium within 5 days, morepreferably within 3 days, from the start of the floating culture, mostpreferably simultaneously with the start of the floating culture. Inaddition, floating culture is performed up to day 18, more preferablyday 12, from the start of the floating culture with the addition of asubstance inhibiting the Wnt signal pathway.

The culture conditions such as culture temperature, and CO₂concentration in the first step can be appropriately determined. Whilethe culture temperature is not particularly limited, it is, for example,about 30 to 40° C., preferably about 37° C. The CO₂ concentration is,for example, about 1 to 10%, preferably about 5%.

The “aggregate” in the present invention refers to a mass of the cellsdispersed in the medium but gathered to form same. The “aggregate” inthe present invention includes an aggregate formed by the cellsdispersed at the start of the floating culture and an aggregate alreadyformed at the start of the floating culture.

When cells are aggregated to form cell aggregates and the aggregates aresubjected to floating culture, to “form aggregate” means to “rapidlyaggregate a given number of dispersed stem cells” to form qualitativelyhomogeneous cell aggregates.

Examples of the experimental operation to form an aggregate include amethod involving keeping cells in a small space by using a plate withsmall wells (96 well plate), micropore and so on, a method involvingaggregating cells by centrifugation for a short time using a smallcentrifugation tube, and so on.

The concentration of the pluripotent stem cells in the first step can bedetermined as appropriate by those of ordinary skill in the art to formaggregates of pluripotent stem cells more uniformly and efficiently. Theconcentration of the pluripotent stem cells when forming aggregates isnot particularly limited as long as it permits formation of uniformaggregates of stem cells. For example, when human ES cells are subjectedto floating culture using a 96 well microwell plate, a liquid preparedto about 1×10³ to about 5×10⁴ cells, preferably about 3×10³ to about3×10⁴ cells, more preferably about 5×10³ to about 2×10⁴ cells, mostpreferably about 9×10³ cells, per well is added, and the plate is leftstanding to form aggregates.

The time of floating culture necessary for forming aggregates can bedetermined as appropriate according to the pluripotent stem cell to beused, as long as the cells can be aggregated rapidly. To form uniformaggregates, it is desirably as short as possible. For example, in thecase of human ES cells, aggregates are desirably formed preferablywithin 24 hr, more preferably within 12 hr. The time for aggregateformation can be appropriately adjusted by those of ordinary skill inthe art by controlling the tools for aggregating the cells,centrifugation conditions and so on.

Those of ordinary skill in the art can determine whether aggregates ofpluripotent stem cells have been formed, based on the size and cellnumber of aggregates, macroscopic morphology, microscopic morphology bytissue staining analysis and uniformity thereof, expression ofdifferentiation and undifferentiation markers and uniformity thereof,control of expression of differentiation marker and synchronism thereof,reproducibility of differentiation efficiency between aggregates, and soon.

(2) Second Step

The second step of subjecting the aggregate formed in the first step tofloating culture in a serum-free medium containing a basement membranepreparation is explained.

The “basement membrane preparation” refers to one containing basementmembrane-constituting components having a function to control cell form,differentiation, growth, motility, expression of function and so onwhich are similar to those of epithelial cell, when intended cellscapable of forming a basement membrane are plated thereon and cultured.Here, the “basement membrane constituting component” refers to anextracellular matrix molecule in the form of a thin membrane presentbetween epithelial cell layer and interstitial cell layer and so on inanimal tissues. A basement membrane preparation can be produced by, forexample, removing cells capable of forming a basement membrane, whichadhere onto a support via a basement membrane, with a solution capableof dissolving the lipid of the cells, an alkali solution and so on.Examples of preferable basement membrane preparation include productscommercially available as basement membrane components (e.g., Matrigel(hereinafter, sometimes referred to as Matrigel)), and extracellularmatrix molecules known as basement membrane components (e.g., laminin,type IV collagen, heparan sulfate proteoglycan, entactin and so on).

Matrigel is a product prepared from a basement membrane derived fromEngelbreth Holm Swarn (EHS) mouse sarcoma. The main component ofMatrigel is type IV collagen, laminin, heparan sulfate proteoglycan, andentactin. In addition to these, TGF-β, fibroblast growth factor (FGF),tissue plasminogen activator, and a growth factor naturally produced byEHS tumor are contained. The “growth factor reduced product” of Matrigelhas a lower growth factor concentration than common Matrigel, and thestandard concentration thereof is <0.5 ng/ml for EGF, <0.2 ng/ml forNGF, <5 pg/ml for PDGF, 5 ng/ml for IGF-1, and 1.7 ng/ml for TGF-β. Inthe method of the present invention, “growth factor reduced product” ispreferably used.

While the concentration of the basement membrane preparation to be addedto a serum-free medium for the floating culture in the second step isnot particularly limited as long as the epithelial structure of theneural tissue (for example, retinal tissue) is stably maintained, forexample, it is preferably 1/20 to 1/200 volume, more preferably about1/100 volume, of the culture medium when Martigel is used. Whilebasement membrane preparation may already have been added to the mediumwhen the culture of stem cell is started, it is preferably added to theserum-free medium within 5 days, more preferably within 2 days, from thestart of the floating culture.

As the serum-free medium to be used in the second step, the serum-freemedium used in the first step may be directly used, or may be replacedwith a fresh serum-free medium.

When the serum-free medium used in the first step is directly used forthis step, the “basement membrane preparation” can be added to themedium.

The serum-free medium used for the floating culture in the first stepand the second step is not particularly limited as long as it is asdefined above. However, to avoid complicated preparation, a serum-freemedium (GMEM or DMEM, 0.1 mM 2-mercaptoethanol, 0.1 mM non-essentialamino acid Mix, 1 mM sodium pyruvate) added with an appropriate amountof commercially available KSR (Knockout Serum Replacement) is preferablyused as the serum-free medium. The amount of KSR to be added to theserum-free medium is not particularly limited and, for example, it isgenerally 1 to 20%, preferably 2 to 20%, in the case of human ES cells.

The culture conditions such as culture temperature, and CO₂concentration in the second step can be appropriately determined. Whilethe culture temperature is not particularly limited, it is, for example,about 30 to 40° C., preferably about 37° C. The CO₂ concentration is,for example, about 1 to 10%, preferably about 5%.

(3) Third Step

The third step of subjecting the aggregate cultured in the second stepto floating culture in a serum-containing medium is explained.

As the serum-containing medium to be used in the third step, may be usedthe serum-free medium used in the culture of the second step to which aserum is directly added, or one replaced with a fresh serum-containingmedium.

As the serum to be added to a medium in the third step, for example,mammalian serum such as bovine serum, calf serum, fetal calf serum,horse serum, colt serum, fetal horse serum, rabbit serum, leveret serum,fetal rabbit serum, and human serum, and so on can be used.

The serum is added on or after day 7, more preferably on or after day 9,most preferably on or after day 12, from the start of the floatingculture. The concentration of the serum to be added is about 1 to 30%,preferably about 3 to 20%, more preferably about 10%.

The serum-containing medium to be used in the third step is notparticularly limited as long as it is as defined above. Theabove-mentioned serum-free medium (GMEM or DMEM, 0.1 mM2-mercaptoethanol, 0.1 mM non-essential amino acid Mix, 1 mM sodiumpyruvate) to which a serum is added is preferably used.

As such serum-containing medium, one to which an appropriate amount ofcommercially available KSR (Knockout Serum Replacement) is added mayalso be used.

In the third step, the production efficiency of retinal tissue can beincreased by adding a substance acting on the Shh signal pathway inaddition to the serum.

The substance acting on the Shh signal pathway is not particularlylimited as long as it can enhance signal transduction mediated by Shh.Examples of the substance acting on the Shh signal pathway includeproteins belonging to the Hedgehog family (e.g., Shh), Shh receptor, Shhreceptor agonist, Putmorphamine, SAG and so on.

The concentration of the substance acting on the Shh signal pathway usedin this step is, for example, in the case of common substance acting onthe Shh signal pathway such as SAG, about 0.1 nM to 10 μM, preferablyabout 10 nM to 1 μM, more preferably about 100 nM.

The thus-produced retinal tissue is present to cover the surface of theaggregate. Whether a retinal tissue is produced by the production methodof the present invention can be confirmed by such immunostaining methodas described in the following (4).

It is also possible to physically cut out the retinal tissue present onthe surface of aggregates with tweezers and so on. In this case, since aneural tissue other than a retinal tissue may be formed on the surfaceof each aggregate, a part of the neural tissue cut out from theaggregate is severed and confirmed by such immunostaining method asdescribed in (4), whereby the tissue is confirmed to be a retinaltissue.

(4) Confirmation Method of Retinal Tissue

A retinal tissue can be produced through the above-mentioned first stepto the third step. Moreover, production of a retinal tissue through thefirst step to the third step can be confirmed by the following method.

The aggregate cultured in the third step is subjected to floatingculture in a serum-containing medium. Examples of the cell culturevessel to be used for floating culture include those mentioned above.The culture conditions such as culture temperature, CO₂ concentration,and O₂ concentration of the floating culture can be appropriatelydetermined. While the culture temperature is not particularly limited,it is, for example, about 30 to 40° C., preferably about 37° C. The CO₂concentration is, for example, about 1 to 10%, preferably about 5%. TheO₂ concentration is, for example, 20 to 70%, preferably 20 to 60%, morepreferably 30 to 50%.

While the culture period in this step is not particularly limited, it isgenerally not less than 48 hr, preferably not less than 7 days.

The retinal tissue can be confirmed by, after completion of the floatingculture, fixing the aggregates with a fixative such as para-formaldehydesolution, preparing a frozen section, and confirming formation of alayer structure by an immunostaining method and so on. Since respectivelayers of a retinal tissue are composed of different retinal progenitorcells (photoreceptor, horizontal cell, bipolar cell, amacrine cell,retinal ganglion cell), formation of a layer structure can be confirmedby an immunostaining method using antibodies against the aforementionedmarkers expressed in these cells.

<Production Method of Optic-Cup-Like Structure>

The second aspect of the present invention is a production method of anoptic-cup-like structure, comprising a step of subjecting the retinaltissue obtained by the above-mentioned <production method of retinaltissue> to floating culture in a serum-free medium or a serum-containingmedium each containing a substance acting on the Shh signal pathway anda substance acting on the Wnt signal pathway. As the retinal tissueobtained in the above-mentioned <production method of retinal tissue>,an aggregate containing the retinal tissue cultured in the third step ofthe above-mentioned <production method of retinal tissue> can be used.As the embodiment of the second invention, a method for producing anoptic-cup-like structure, comprising the following steps (1) to (4) canbe mentioned:

(1) a first step of subjecting pluripotent stem cells to floatingculture in a serum-free medium containing a substance inhibiting the Wntsignal pathway to form an aggregate of pluripotent stem cells,(2) a second step of subjecting the aggregate formed in the first stepto floating culture in a serum-free medium containing a basementmembrane preparation,(3) a third step of subjecting the aggregate cultured in the second stepto floating culture in a serum-containing medium, and(4) a fourth step of subjecting the aggregate cultured in the third stepto floating culture in a serum-free medium or serum-containing mediumeach containing a substance acting on the Shh signal pathway and asubstance acting on the Wnt signal pathway.

Here, the substance acting on the Shh signal pathway is not particularlylimited as long as it can enhance signal transduction mediated by Shh.Examples of the substance acting on the Shh signal pathway includeproteins belonging to Hedgehog family (e.g., Shh), Shh receptor, Shhreceptor agonist, Purmorphamine, SAG and so on.

The concentration of the substance acting on the Shh signal pathway tobe used in the second aspect of the present invention is, for example,in the case of common substance acting on the Shh signal pathway such asSAG, about 0.1 nM to 10 μM, preferably about 10 nM to 1 μM, morepreferably about 100 nM.

Examples of the substance acting on the Wnt signal pathway includeprotein belonging to Wnt family, Wnt receptor, Wnt receptor agonist,GSK3β inhibitor (e.g., 6-Bromoindirubin-3′-oxime (BIO), CHIR99021,Kenpaullone) and so on.

The concentration of the substance acting on the Wnt signal pathway tobe used in the second aspect of the present invention is, for example,in the case of common substance acting on the Wnt signal pathway such asCHIR99021, about 0.1 μM to 100 μM, preferably about 1 μM to 30 μM, morepreferably about 3 μM.

The substance acting on the Shh signal pathway and the substance actingon the Wnt signal pathway are added on or after day 12 and on or beforeday 25, preferably on or after day 15 and on or before day 18, from thestart of the floating culture. In this case, a medium free of thesubstance inhibiting the Wnt signal pathway added in the aggregateformation step is preferably used.

An optic-cup-like structure is produced in the form of a protrusion froman aggregate on or after day 18 from the start of the floating culture.Whether it is an optic-cup-like structure can be confirmed by those ofordinary skill in the art by observation with a microscope, a magnifyingglass and so on.

The thus-produced optic-cup-like structure is formed in a two-layerstructure of outer layer and inner layer. Since retinal pigmentepitheliua are present in the outer layer and the retinal progenitorcells are present in the inner layer, the retinal progenitor cell andthe retinal pigment epithelium can be observed by, for example,preparing a frozen section of the optic-cup-like structure andperforming immunostaining.

Furthermore, since the optic-cup-like structure produced by the methodof the present invention is formed in the form of a protrusion from anaggregate, it is also possible to obtain a highly pure retinalprogenitor cell by physically and morphologically cutting out theprotrusion from the aggregate, followed by applying the resultingoptic-cup-like structures to a dispersion treatment (e.g., trypsin/EDTAtreatment) and FACS sorting. The method for cutting out theoptic-cup-like structure is not particularly limited, and it can be cutout easily from an aggregate of stem cells using fine tweezers and soon.

<Production Method of Retinal Layer-Specific Neural Cell>

The third aspect of the present invention is a method of producing aretinal layer-specific neural cell, comprising bringing a retinalprogenitor cell contained in a retinal tissue derived from a primatepluripotent stem cell into contact with a substance inhibiting the Notchsignal pathway. According to the method of the present invention, aretinal layer-specific neural cell can be produced from a retinalprogenitor cell.

(Production Method of Primate Pluripotent Stem Cell-Derived RetinalTissue)

The “primate pluripotent stem cell-derived retinal tissue” used in theproduction method of the retinal layer-specific neural cell isexplained.

As a primate pluripotent stem cell-derived retinal tissue, for example,the retinal tissue obtained by the above-mentioned <production method ofretinal tissue>, or the retinal tissue produced from the optic-cup-likestructure obtained by the above-mentioned <production method ofoptic-cup-like structure> can be used.

In the latter case, the retinal tissue can be produced by subjecting theoptic-cup-like structure formed in the fourth step of theabove-mentioned <production method of optic-cup-like structure> to afurther floating culture.

Since an optic-cup-like structure is formed in the form of a protrusionfrom an aggregate, as mentioned above, a highly pure retinal tissue canbe obtained by physically and morphologically cutting out the protrusionfrom the aggregate, followed by separation and culturing. The method forcutting out the optic-cup-like structure is not particularly limited,and it can be cut out easily from an aggregate of stem cells using finetweezers and so on.

The retinal tissue and the optic-cup-like structure produced asmentioned above contain retinal progenitor cells, and retinallayer-specific neural cells can be produced from retinal progenitorcells by bringing the aforementioned retinal progenitor cells intocontact with a substance inhibiting the Notch signal pathway.

<Substance Inhibiting the Notch Signal Pathway>

Next, the substance inhibiting Notch signal pathway used in theproduction method of the retinal layer-specific neural cell isexplained.

The substance inhibiting Notch signal pathway is not particularlylimited as long as it can inhibit signal transduction mediated by Notch.Examples of the substance inhibiting the Notch signal pathway includeNotch antibody, Notch receptor antagonist, ADAM inhibitor, gammasecretase inhibitor and so on.

Examples of the gamma secretase inhibitor includeN—[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPI).

The concentration of the gamma secretase inhibitor is not particularlylimited as long as it can enhance differentiation of retinal progenitorcell into photoreceptor precursor cell or photoreceptor. Theconcentration is, for example, in the case of common gamma secretaseinhibitors, about 0.1 to 1000 μM, preferably about 1 to 100 μM, morepreferably about 10 μM.

The gamma secretase inhibitor is added on or after day 15 and on orbefore day 200 from the start of the floating culture of primatepluripotent stem cells, for the retinal tissues produced from primatepluripotent stem cells. Preferably, a gamma secretase inhibitor is addedto the medium on or after day 20 and on or before day 150, morepreferably on or after day 25 and on or before day 100, ofdifferentiation induction.

The period of the adhesion culture in the presence of the gammasecretase inhibitor can be a length which allow more efficientproduction of the photoreceptor precursor cell or photoreceptor. Thelength of such period can be, for example, about 3 days or more,preferably about 5 to 100 days, more preferably about 7 to 30 days.

(Confirmation Method of Retinal Layer-Specific Neural Cell)

A method for confirming a retinal layer-specific neural cell produced asmentioned above is explained by referring to the case where the retinallayer-specific neural cell is a photoreceptor, a photoreceptorprecursor, or a ganglion cell.

Whether the produced retinal layer-specific neural cell is aphotoreceptor precursor cell can be confirmed by a known method, forexample, expression of a photoreceptor precursor cell marker. Examplesof the photoreceptor precursor cell marker include Crx.

Photoreceptor contains rod cell and cone cell. Whether the produced cellis a photoreceptor can be confirmed by a method known per se, forexample, expression of a photoreceptor marker. Examples of thephotoreceptor marker include rhodopsin (rod cell), red/green opsin (conecell), blue opsin (cone cell), recoverin (rod cell, cone cell) and soon.

In addition, whether the produced retinal layer-specific neural cell isa ganglion cell can be confirmed by a known method, for example,expression of ganglion cell marker. Examples of the ganglion cell markerinclude Brn3.

After completion of the adhesion culture, photoreceptor precursor cellor photoreceptor can be isolated from the retinal tissue. Such isolationcan be performed by a method known per se (cell sorter etc.) and usingan antibody against the surface marker of a photoreceptor precursor cellor photoreceptor and so on. In addition, after completion of theculture, a ganglion cell can be isolated from the retinal tissue. Suchisolation can be performed by a method known per se (cell sorter etc.)and using an antibody against the surface marker of a ganglion cell andso on. Alternatively, by using, as a pluripotent stem cell, a cellwherein a labeled gene (e.g., fluorescent protein such as GFP) has beenknocked in-frame in a gene encoding a marker (e.g., Crx) of aphotoreceptor precursor cell or a marker (e.g., recoverin) of aphotoreceptor or a marker (Brn3) of a ganglion cell, each cell can beisolated by a method known per se (cell sorter etc.) using theexpression of the label gene as an indicator.

<Production Method of Retinal Pigment Epithelium>

The fourth aspect of the present invention is a production method of aretinal pigment epithelium, comprising a step of subjecting the retinaltissue obtained by the above-mentioned <production method of retinaltissue> to floating culture in a serum-free medium or a serum-containingmedium each containing a substance acting on the Wnt signal pathway (butnot containing a substance acting on the Sonic hedgehog signal pathway).As the retinal tissue obtained in the above-mentioned <production methodof retinal tissue>, an aggregate containing the retinal tissue culturedin the third step of the above-mentioned <production method of retinaltissue> can be used. As the embodiment of the fourth invention, a methodof producing a retinal pigment epithelium, comprising the followingsteps (1) to (4) can be mentioned:

(1) a first step of subjecting pluripotent stem cells to floatingculture in a serum-free medium containing a substance inhibiting the Wntsignal pathway to form an aggregate of pluripotent stem cells,(2) a second step of subjecting the aggregate formed in the first stepto floating culture in a serum-free medium containing a basementmembrane preparation,(3) a third step of subjecting the aggregate cultured in the second stepto floating culture in a serum-containing medium, and(4) a fourth step of subjecting the aggregate cultured in the third stepto floating culture in a serum-free medium or serum-containing mediumeach containing a substance acting on the Wnt signal pathway, whereinthe aforementioned serum-free medium and serum-containing medium arefree of a substance acting on the Sonic hedgehog signal pathway.

Here, examples of the substance acting on the Wnt signal pathway includeproteins belonging to Wnt family, Wnt receptor, Wnt receptor agonist,GSK3β inhibitor (e.g., 6-Bromoindirubin-3′-oxime (BIO), CHIR99021,Kenpaullone) and so on.

The concentration of the substance acting on the Wnt signal pathway tobe used in the fourth aspect of the present invention is, for example,in the case of common substance acting on the Wnt signal pathway such asCHIR99021, about 0.1 μM to 100 μM, preferably about 1 μM to 30 μM, morepreferably about 3 μM.

The substance acting on the Wnt signal pathway is added on or after day12, most preferably on day 15, when, for example, human ES cells areused. In this case, preferably used is a medium free of the substanceinhibiting the Wnt signal pathway added in the first step and thesubstance acting on the Shh signal pathway added in the third step.

In the fourth aspect of the present invention, the retinal tissue or theaggregate containing a retinal tissue, which is obtained by theabove-mentioned <production method of retinal tissue>, is preferablycultured in a serum-free medium or a serum-containing medium eachcontaining a substance acting on the Wnt signal pathway and a substanceacting on the Activin signal pathway.

The substance acting on the Activin signal pathway is not particularlylimited as long as it can enhance signal transduction mediated byActivin. Examples of the substance acting on the Activin signal pathwayinclude proteins belonging to the Activin family (e.g., Activin A,Activin B, Activin C, and Activin AB, etc.), Activin receptor, Activinreceptor agonist and so on.

The concentration of the substance acting on the Activin signal pathwayto be used in this step is, for example, in the case of common substanceacting on the Activin signal pathway such as Recombinant Human/Mouse/RatActivin A (R&D systems #338-AC), 1 ng/ml to 10 ug/ml, preferably about10 ng/ml to 1 ug/ml, more preferably about 100 ng/ml.

Since the thus-produced retinal pigment epithelium is present on thesurface of aggregates, it can be easily observed by microscopicobservation and so on. It is also possible to obtain a highly pureretinal pigment epithelium by subjecting an aggregate containing theretinal pigment epithelium to, for example, a dispersion treatment(e.g., trypsin/EDTA treatment) followed by FACS sorting. It is alsopossible to physically cut out the retinal pigment epithelium from theaggregates with tweezers and so on, followed by cultivation. The retinalpigment epithelium after dispersion or cutting out can be cultured underadhesion conditions. In the case of adhesion culture, a cell adhesiveculture vessel, for example, a culture vessel after a coating treatmentwith an extracellular matrix etc. (e.g., poly-D-lysine, laminin,fibronectin), is preferably used. The culture conditions of the adhesionculture such as culture temperature, CO₂ concentration, and O₂concentration can be easily determined by those of ordinary skill in theart. In this case, culture may be performed in the presence of a serum,a known growth factor, an additive and a chemical substance that promotethe growth. Examples of the known growth factor include EGF, FGF and soon. Examples of the additive that promotes the growth include N2supplement (Invitrogen), B27 supplement (Invitrogen) and so on.

<Use of Retinal Tissue as a Reagent for Evaluating Toxicity or DrugEfficacy>

The retinal tissue, optic-cup-like structure, retinal layer-specificneural cell and retinal pigment epithelium produced by the first to thefourth aspects of the present invention can also be used for screeningfor a therapeutic drug for a disease due to a disorder of retinal tissueor retina-related cell, or a transplantation material for celltreatment, a material for the study of diseases or a drug discoverymaterial for a therapeutic drug for a cell damage due to other etiology.In addition, they can be utilized for the study, test and so on of suchtoxicity as phototoxicity in the toxicity and drug efficacy evaluationof chemical substances and so on.

Examples of the disease due to a disorder of retinal tissue orretina-related cell include organic mercury poisoning, chloroquineretinopathy, retinitis pigmentosa, age-related macular degeneration,glaucoma, diabetic retinopathy, neonatal retinopathy, and so on.

<Use of Retinal Tissue, Optic-Cup-Like Structure, Retinal Layer-SpecificNeural Cell and Retinal Pigment Epithelium as Biological Materials forTransplantation>

The retinal tissue, optic-cup-like structure, retinal layer-specificneural cell and retinal pigment epithelium produced by the first to thefourth aspects of the present invention can be used as biologicalmaterials for transplantation used for supplementing a damaged cell ordisordered tissue itself in a cell damage state (e.g., used fortransplantation operation) and so on. Examples of the transplantationmethod include, but are not limited to, the methods described in thebelow-mentioned Examples.

The production method of the present invention is explained in moredetail in the following by referring to Comparative Examples andExamples. The Examples merely show exemplification of the presentinvention and do not limit the scope of the present invention in anyway.

EXAMPLES Establishment of RAX Knock-in Human ES Cell

Human ES cell line with GFP knocked-in at the RAX gene locus, which isone of the marker genes of retinal progenitor cell, was produced.

Zinc Finger Nuclease (ZFN) that specifically cleaves RAX gene on genomicDNA of human ES cell line (KhES-1: human ES cell line established byKyoto University) was purchased from Sigma-Aldrich Co. LLC. Using humanES cells which were dissociated to single cells and according to theelectroporation method, ZFN-coding mRNA and a knock-in vector carryingGFP and a neomycin-resistance gene, which is a drug selection gene, wereco-transfected, and plated on neomycin resistance mouse fibroblasttreated with mitomycin C. From the next day of plating, G418 was addedinto the medium and drug selection was performed. The colony of theobtained resistant clone was picked up, culture was continued, andknock-in cells were selected by the PCR method and the Southern blotmethod, whereby a RAX::GFP-knock-in human ES cell line was established.

Comparative Example 1 Production of Retinal Tissue by Using Human ESCells (Matrigel Addition Conditions)

RAX::GFP knock-in human ES cells (derived from KhES-1) were culturedaccording to the methods described in “Ueno, M. et al. PNAS 2006”,“Watanabe, K. et al. Nat Biotech 2007” and used for the experiment. Asthe medium, DMEM/F12 medium (Invitrogen) added with 20% KSR (KnockoutSerum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 1 mM pyruvicacid, and 5 to 10 ng/ml bFGF was used. The ES cells were dispersed intosingle cells by using 0.25% trypsin-EDTA (Invitrogen), andfloating-cultured in a serum-free medium (100 μl) at 37° C., 5% CO₂ to9×10̂3 cells per well of a non-cell adhesive 96-well culture plate(SUMILON spheroid plate, SUMITOMO BAKELITE CO., LTD.). As the serum-freemedium in this case, a serum-free medium obtained by adding 20% KSR, 0.1mM 2-mercaptoethanol, 1 mM pyruvic acid and 20 μM Y27632 to G-MEM mediumwas used. During the floating culture, Matrigel in an amount of 1/100per volume was added from day 2 from the start of the floating culture.Thereafter, fluorescence microscopic observation was regularly performedwhile continuing the floating culture.

As a result of the fluorescence microscopic observation up to day 25from the start of the floating culture, GFP expression cells showinginduction of the retinal progenitor cells were somewhat found (FIGS. 1A,B).

Comparative Example 2 Production of Retinal Tissue by Using Human ESCells (Nodal Signal Pathway Inhibitor and Matrigel Addition Conditions)

RAX::GFP knock-in human ES cells (derived from KhES-1) were culturedaccording to the methods described in “Ueno, M. et al. PNAS 2006”,“Watanabe, K. et al. Nat Biotech 2007” and used for the experiment. Asthe medium, DMEM/F12 medium (Invitrogen) added with 20% KSR (KnockoutSerum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 1 mM pyruvicacid, and 5-10 ng/ml bFGF was used. For the production of retinal tissueby floating culture, the ES cells were dissociated into single cells byusing 0.25% trypsin-EDTA (Invitrogen), and floating-cultured in aserum-free medium (100 μl) at 37° C., 5% CO₂ to 9×10̂3 cells per well ofa non-cell adhesive 96-well culture plate (SUMILON spheroid plate,SUMITOMO BAKELITE CO., LTD.). As the serum-free medium in this case, aserum-free medium obtained by adding 20% KSR, 0.1 mM 2-mercaptoethanol,1 mM pyruvic acid, 20 μM Y27632 and a substance inhibiting the Nodalsignal pathway (10 μM SB431542) to G-MEM medium was used. During thefloating culture, Matrigel in an amount of 1/100 per volume was addedfrom day 2 from the start of the floating culture. Thereafter, thefloating culture was continued and fluorescence microscopic observationand confirmation of the proportion of the GFP-expressing cells wereperformed by FACS on day 18 from the start of the floating culture.

As a result, GFP-expressing cells were somewhat found (FIGS. 10, D).

Comparative Example 3 Production of Retinal Tissue by Using Human ESCells (Wnt Signal Pathway Inhibitor and Matrigel Addition Conditions)

RAX::GFP knock-in human ES cells (derived from KhES-1) were culturedaccording to the methods described in “Ueno, M. et al. PNAS 2006”,“Watanabe, K. et al. Nat Biotech 2007” and used for the experiment. Asthe medium, DMEM/F12 medium (Invitrogen) added with 20% KSR (KnockoutSerum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 1 mM pyruvicacid, and 5 to 10 ng/ml bFGF was used. For the production of retinaltissue by floating culture, the ES cells were dispersed into singlecells by using 0.25% trypsin-EDTA (Invitrogen), and floating-cultured ina serum-free medium (100 μl) at 37° C., 5% CO₂ to 9×10̂3 cells per wellof a non-cell adhesive 96-well culture plate (SUMILON spheroid plate,SUMITOMO BAKELITE CO., LTD.). As the serum-free medium in this case, aserum-free medium obtained by adding 20% KSR, 0.1 mM 2-mercaptoethanol,1 mM pyruvic acid, 20 μM Y27632 and a substance inhibiting the Wntsignal pathway (3 μM IWR1e) to G-MEM medium was used. During thefloating culture, Matrigel in an amount of 1/100 per volume was addedfrom day 2 from the start of the floating culture. Thereafter, thefloating culture was continued and fluorescence microscopic observationand confirmation of the proportion of the GFP-expressing cells wereperformed by FACS on day 18 from the start of the floating culture.

As a result, the GFP-expressing cells clearly increased (FIGS. 1E, F) ascompared to Comparative Examples 1 and 2.

Example 1 Production of Retinal Tissue by Using Human ES Cells (WntSignal Pathway Inhibitor and Matrigel, Serum Addition Conditions)

RAX::GFP knock-in human ES cells (derived from KhES-1) were culturedaccording to the methods described in “Deno, M. et al. PNAS 2006”,“Watanabe, K. et al. Nat Biotech 2007” and used for the experiment. Asthe medium, DMEM/F12 medium (Invitrogen) added with 20% KSR (KnockoutSerum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 1 mM pyruvicacid, and 5 to 10 ng/ml bFGF was used. For the production of retinaltissue by floating culture, the ES cells were dispersed into singlecells by using 0.25% trypsin-EDTA (Invitrogen), and floating-cultured ina serum-free medium (100 μl) at 37° C., 5% CO₂ to 9×10̂3 cells per wellof a non-cell adhesive 96-well culture plate (SUMILON spheroid plate,SUMITOMO BAKELITE CO., LTD.). As the serum-free medium in this case, aserum-free medium obtained by adding 20% KSR, 0.1 mM 2-mercaptoethanol,1 mM pyruvic acid, 20 μM Y27632 and a substance inhibiting the Wntsignal pathway (3 μM IWR1e) to G-MEM medium was used. During thefloating culture, Matrigel in an amount of 1/100 per volume was addedfrom day 2 from the start of the floating culture. Furthermore, a fetalcalf serum in a 1/10 amount per volume was added on day 12 from thestart of the floating culture. Thereafter, the floating culture wascontinued and fluorescence microscopic observation and confirmation ofthe proportion of the GFP-expressing cells were performed by FACS on day18 from the start of the floating culture. Simultaneously, an experimentwas also performed under the conditions of Comparative Example 3, whichwere free of the addition of a serum.

While the proportion of the GFP-expressing cells under the conditions ofComparative Example 3 was 3.2% (FIGS. 2A, B, FIG. 3A), manyGFP-expressing cells emerged under the conditions with the addition of aserum (FIGS. 2C, D). The proportion of the GFP-positive cells was over30% in the analysis by FACS (FIG. 3B).

Example 2 Production of Retinal Tissue by Using Human ES Cells(Conditions with Addition of Wnt Signal Pathway Inhibitor and Matrigel,Serum and a Substance Acting on the Shh Signal)

RAX::GFP knock-in human ES cells (derived from KhES-1) were culturedaccording to the methods described in “Ueno, M. et al. PNAS 2006”,“Watanabe, K. et al. Nat Biotech 2007” and used for the experiment. Asthe medium, DMEM/F12 medium (Invitrogen) added with 20% KSR (KnockoutSerum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 1 mM pyruvicacid, and 5 to 10 ng/ml bFGF was used. For the production of retinaltissue by floating culture, the ES cells were dispersed into singlecells by using 0.25% trypsin-EDTA (Invitrogen), and floating-cultured ina serum-free medium (100 μl) at 37° C., 5% CO₂ to 9×10̂3 cells per wellof a non-cell adhesive 96-well culture plate (SUMILON spheroid plate,SUMITOMO BAKELITE CO., LTD.). As the serum-free medium in this case, aserum-free medium obtained by adding 20% KSR, 0.1 mM 2-mercaptoethanol,1 mM pyruvic acid, 20 μM Y27632 and a substance inhibiting the Wntsignal pathway (3 μM IWR1e) to G-MEM medium was used. During thefloating culture, Matrigel in an amount of 1/100 per volume was addedfrom day 2 from the start of the floating culture. A fetal calf serum inan amount of 1/10 per volume and a substance acting on the Shh signalpathway (100 nM SAG) were added on day 12 from the start of the floatingculture. The proportion of the GFP-expressing cells was measured by FACSon day 18 from the start of the floating culture.

When a substance acting on the Shh signal pathway was addedsimultaneously with a serum, a very high number of GFP-expressing cellsemerged (FIGS. 2E, F). From the analysis using FACS, the proportion ofthe GFP-expressing cells was found to have reached not less than 70%.

It was found that, as compared to Comparative Example 3, the proportionof the GFP-expressing cells increased to about 10-fold under theconditions of Example 1 wherein a serum was added, and further, theproportion of the GFP-expressing cells increased to about 24-fold underthe conditions of Example 2 wherein a serum and a substance acting onthe Shh signal pathway were simultaneously added (FIG. 3D).

Example 3 Confirmation of Retinal Tissue Formation (Method)

Formation of retinal tissue was confirmed with the aggregates havingGFP-expressing cells, which were produced in Examples 2 and 3. Using aserum-containing medium obtained by adding N2 supplement, 10% (v/v)fetal calf serum and 0.5 μM retinoic acid to DMEM/F12 medium, floatingculture was performed under the conditions of 40% O₂ from day 18 fromthe start of the floating culture. Thereafter, the aggregated mass wasfixed with 4% para-formaldehyde solution, a frozen section was prepared,and the tissue structure was confirmed by the immunostaining method.

As a result, it was revealed on day 60 from the start of the floatingculture that Brn3 and TuJ1-positive ganglion cells in the lowermostlayer, Crx and Recoverin-positive photoreceptor-precursor cells in theoutermost layer and intermediate layer, and interneuron progenitor cellssuch as Chx10-positive bipolar cells between the Brn3-positive cells inthe outermost layer and the Brn3-positive cells in the lowermost layerwere arranged in layers in an orderly manner (FIGS. 4A, B, C).Furthermore, when the floating culture was continued until day 126, Crxand Recoverin-positive photoreceptor-precursor cells were accumulated inthe outermost layer, and the cells expressing Nrl that is specificallyexpressed in rod cells and the cells expressing Rxr-gamma that isspecifically expressed in cone cells were observed. In addition, thecells expressing Ptf1a, which is a precursor cell marker of horizontalcell and amacrine cell, were observed in the intermediate layer (FIGS.4D, E, F, G, H, I). From these results, it has been shown that a retinaltissue can be produced at high efficiency from human ES cells.

Example 4 Transplantation into Eye of Retinal Tissue Produced from HumanES Cell

After incision of the sclera of an eyeball, an injection needle wasinserted from the sclera incision into the vitreous to lower theintraocular pressure. An intraocular perfusion fluid was injected fromthe sclera incision into subretinal space with a cell transplantationneedle to artificially form a shallow retinal detachment state. Theretinal tissue is transplanted with a cell transplantation needle or acell sheet transplantation device into the space formed.

Example 5 Production of Optic-Cup-Like Structure at High EfficiencyUsing Human ES Cell (Method)

Using RAX::GFP knock-in human ES cells, an optic-cup-like structure wasproduced.

RAX::GFP knock-in human ES cells (derived from KhES-1) were culturedaccording to the methods described in “Ueno, M. et al. PNAS 2006”,“Watanabe, K. et al. Nat Biotech 2007” and used for the experiment. Asthe medium, DMEM/F12 medium (Invitrogen) added with 20% KSR (KnockoutSerum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 1 mM pyruvicacid, and 5 to 10 ng/ml bFGF was used. For the formation of aggregate byfloating culture, the ES cells were dispersed into single cells by using0.25% trypsin-EDTA (Invitrogen), suspended in a serum-free medium (100μl) to 9×10̂3 cells per well of a non-cell adhesive 96-well culture plate(SUMILON spheroid plate, SUMITOMO BAKELITE CO., LTD.) to allow for rapidformation of aggregates, and floating cultured at 37° C., 5% CO₂. As theserum-free medium in this case, a serum-free medium obtained by adding20% KSR, 0.1 mM 2-mercaptoethanol, 1 mM pyruvic acid, 20 μM Y27632 and asubstance inhibiting the Wnt signal pathway (3 μM IWR1e) to G-MEM mediumwas used. During the floating culture, Matrigel in an amount of 1/100per volume was added from day 2 from the start of the floating culture.The floating aggregate was transferred to a serum-free medium without asubstance inhibiting the Wnt signal pathway on day 12 from the start ofthe floating culture, fetal calf serum in an amount of 1/10 per volumewas added and the aggregate was cultured. Furthermore, the floatingculture was performed in a serum-containing medium containing asubstance acting on the Wnt signal pathway (3 μM CHIR99021) and asubstance acting on the Shh signal pathway (100 nM SAG) from day 15.

(Results)

When produced by the above-mentioned method, a GFP-positive cellpopulation showing expression of RAX emerged in a part of the aggregatearound day 14 from the start of the floating culture (FIG. 5A), whichthen raised toward the outer side of the aggregate (FIGS. 5B, C). Whenthe floating culture was continued, a clear protrusion was formed (FIG.5D). Furthermore, when the floating culture was continued, a clearoptic-cup-like structure was formed on the aggregate composed of theGFP-positive cells (retinal progenitor cells) (FIG. 6). Thisoptic-cup-like structure could be clearly recognized simply bymicroscopic observation of the aggregate (FIG. 6A). When theoptic-cup-like structure formed on the aggregate was observed in depthby a two-photon microscope to find that it had a two-layer structure ofthe outside and the inside (FIGS. 6C, D). A frozen section of theoptic-cup-like structure was prepared and immunostained. As a result, alayer of retinal pigment epithelia expressing Mitf was present on theoutside, and Chx10-positive neural retinal progenitor cells were presentin the inside thereof (FIG. 7).

Example 6 Transplantation into Eye of Optic-Cup-Like Structure Producedfrom Human ES Cell

After incision of the sclera of an eyeball, an injection needle wasinserted from the sclera incision into the vitreous to lower theintraocular pressure. An intraocular perfusion fluid was injected fromthe sclera incision into subretinal space with a cell transplantationneedle to artificially form a shallow retinal detachment state. Thecultured optic-cup-like structure is transplanted with a celltransplantation needle or a cell sheet transplantation device into thespace formed.

Example 7 Treatment of Human ES Cell-Derived Retinal Tissue with aSubstance Acting on the Notch Signal

CrX::GFP knock-in human ES cells (derived from KhES-1) were culturedaccording to the methods described in “Ueno, M. et al. PNAS 2006”,“Watanabe, K. et al. Nat Biotech 2007” and used for the experiment. Asthe medium, DMEM/F12 medium (Invitrogen) added with 20% KSR (KnockoutSerum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 1 mM pyruvicacid, and 5 to 10 ng/ml bFGF was used. The ES cells were dispersed intosingle cells by using 0.25% trypsin-EDTA (Invitrogen), andfloating-cultured in a serum-free medium (100 μl) at 37° C., 5% CO₂ to9×10̂3 cells per well of a non-cell adhesive 96-well culture plate(SUMILON spheroid plate, SUMITOMO BAKELITE CO., LTD.). As the serum-freemedium in this case, a serum-free medium obtained by adding 20% KSR, 0.1mM 2-mercaptoethanol, 1 mM pyruvic acid, 20 μM Y27632 and a substanceinhibiting the Wnt signal pathway (3 μM IWR1e) to G-MEM medium was used.Matrigel was added in an amount of 1/100 per volume from day 2 of thefloating culture and floating culture was performed. Fetal calf serum inan amount of 1/10 per volume and a substance acting on the Shh signalpathway (100 nM SAG) were added on day 12 of floating culture and thefloating culture was performed to produce a retinal tissue. A substanceacting on the Notch signal (10 μM DAPT (gamma secretase activityinhibitor)) was added to the retinal tissue on day 29 from the start ofthe floating culture, observed with a fluorescence microscope on day 41from the start of the floating culture (day 12 after the addition), andfixed with 4% para-formaldehyde on day 43 from the start of the floatingculture (day 14 after the addition), and a frozen section was prepared.The prepared frozen section was immunostained for Recoverin, which isone of the marker genes of photoreceptor, and Brn3, which is one of themarker genes of ganglion cells, and the results were compared betweenthe presence and absence of DAPT-addition.

As a result, the GFP-expressing cells markedly increased when DAPT wasadded (FIG. 8B), as compared to when DAPT was not added (FIG. 8A). Inaddition, from the results of immunostaining of the frozen section, itwas revealed that Recoverin-positive cells increased 3- to 5-fold whenDAPT was added (FIGS. 8C, D).

These results show a marked increase in the photoreceptors. In addition,it was found from the results of immunostaining of Brn3 that theganglion cells also increased by the addition of DAPT (FIGS. 8E, F).

Example 8 Treatment of Human ES Cell-Derived Retinal Tissue afterFreeze-Thawing with a Substance Acting on the Notch Signal

RAX::GFP knock-in human ES cells (derived from KhES-1) were culturedaccording to the methods described in “Deno, M. et al. PNAS 2006”,“Watanabe, K. et al. Nat Biotech 2007” and used for the experiment. Asthe medium, DMEM/F12 medium (Invitrogen) added with 20% KSR (KnockoutSerum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 1 mM pyruvicacid, and 5 to 10 ng/ml bFGF was used. For the production of retinaltissue by floating culture, the ES cells were dispersed into singlecells by using 0.25% trypsin-EDTA (Invitrogen), and floating-cultured ina serum-free medium (100 μl) at 37° C., 5% CO₂ to 9×10̂3 cells per wellof a non-cell adhesive 96-well culture plate (SUMILON spheroid plate,SUMITOMO BAKELITE CO., LTD.). As the serum-free medium in this case, aserum-free medium obtained by adding 20% KSR, 0.1 mM 2-mercaptoethanol,1 mM pyruvic acid, 20 μM Y27632 and a substance inhibiting the Wntsignal pathway (3 μM IWR1e) to G-MEM medium was used. A serum-freemedium added with Matrigel in an amount of 1/100 per volume was usedfrom day 2 from the start of the floating culture. Matrigel was added inan amount of 1/100 per volume from day 2 of the floating culture andfloating culture was performed. Fetal calf serum in an amount of 1/10per volume and a substance acting on the Shh signal pathway (100 nM SAG)were added on day 12 from the start of the floating culture and thefloating culture was performed to produce a retinal tissue. The producedretinal tissue on day 33 from the start of the floating culture wasfreeze-thawed, and a Notch signal action inhibitory substance (10 μMDAPT) was added to the retinal tissue on day 38 from the start of thefloating culture. The tissue was observed with a fluorescence microscopeon day 49 from the start of the floating culture (day 11 after theaddition), and fixed with 4% para-formaldehyde, and a frozen section wasprepared. The prepared frozen section was immunostained for Recoverin,which is one of the marker genes of photoreceptor, and Brn3, which isone of the marker genes of ganglion cells, and the results were comparedbetween the presence and absence of DAPT-addition.

As a result, the GFP-expressing cells were markedly produced when DAPTwas added (FIG. 9B), as compared to when DAPT was not added (FIG. 9A).In addition, from the results of immunostaining of the frozen section,it was revealed that Recoverin-positive cells were produced about 5-foldwhen DAPT was added (FIGS. 9C, D). These results show a markedproduction of the photoreceptors. In addition, it was found from theresults of immunostaining of Brn3 that the ganglion cells were alsoproduced by the addition of DAPT (FIGS. 9E, F).

Example 9 Transplantation into Eye of Retinal Layer-Specific Neural CellProduced from Human ES Cell

After incision of the sclera of an eyeball, an injection needle wasinserted from the sclera incision into the vitreous to lower theintraocular pressure. An intraocular perfusion fluid was injected fromthe sclera incision into subretinal space with a cell transplantationneedle to artificially form a shallow retinal detachment state. Theretinal layer-specific neural cell is transplanted with a celltransplantation needle or a cell sheet transplantation device into thespace formed.

Example 10 Production of Retinal Pigment Epithelium at High EfficiencyUsing Human ES Cell (Method)

Human ES cells (KhES-1) were cultured according to the methods describedin “Ueno, M. et al. PNAS 2006”, “Watanabe, K. et al. Nat Biotech 2007”and used for the experiment. As the medium, DMEM/F12 medium (Invitrogen)added with 20% KSR (Knockout Serum Replacement; Invitrogen), 0.1 mM2-mercaptoethanol, 1 mM pyruvic acid, and 5 to 10 ng/ml bFGF was used.For the formation of retinal tissue by floating culture, the ES cellswere dispersed into single cells by using 0.25% trypsin-EDTA(Invitrogen), suspended in a serum-free medium (100 μl) to 9×10̂3 cellsper well of a non-cell adhesive 96-well culture plate (SUMILON spheroidplate, SUMITOMO BAKELITE CO., LTD.) to allow for rapid formation ofaggregates, and floating cultured at 37° C., 5% CO₂. As the serum-freemedium in this case, a serum-free medium obtained by adding 20% KSR, 0.1mM 2-mercaptoethanol, 1 mM pyruvic acid, 20 μM Y27632 and a substanceinhibiting the Wnt signal pathway (3 μM IWR1e) to G-MEM medium was used.Matrigel was added in an amount of 1/100 per volume from day 2 of thefloating culture and floating culture was performed. The aggregate wastransferred to a serum-free medium without containing a substanceinhibiting the Wnt signal pathway on day 12 from the start of thefloating culture, fetal calf serum in an amount of 1/10 per volume wasadded and cultured. The floating culture was performed in a mediumcontaining a substance acting on the Wnt signal pathway (3 μM CHIR99021)from day 15.

(Results)

When produced by the above-mentioned method, a retinal pigmentepithelium population emerged on almost all surfaces of the aggregates(FIG. 10).

Example 11 Production of Retinal Pigment Epithelium Using Human ES Cell(Method)

Human ES cells (KhES-1) were cultured according to the methods describedin “Ueno, M. et al. PNAS 2006”, “Watanabe, K. et al. Nat Biotech 2007”and used for the experiment. As the medium, DMEM/F12 medium (Invitrogen)added with 20% KSR (Knockout Serum Replacement; Invitrogen), 0.1 mM2-mercaptoethanol, 1 mM pyruvic acid, and 5 to 10 ng/ml bFGF was used.For the formation of retinal tissue by floating culture, the ES cellswere dispersed into single cells by using 0.25% trypsin-EDTA(Invitrogen), suspended in a serum-free medium (100 μl) to 9×10̂3 cellsper well of a non-cell adhesive 96-well culture plate (SUMILON spheroidplate, SUMITOMO BAKELITE CO., LTD.) to allow for rapid formation ofaggregates, and floating cultured at 37° C., 5% CO₂. As the serum-freemedium in this case, a serum-free medium obtained by adding 20% KSR, 0.1mM 2-mercaptoethanol, 1 mM pyruvic acid, 20 μM Y27632 and a substanceinhibiting the Wnt signal pathway (3 μM IWR1e) to G-MEM medium was used.During the floating culture, Matrigel in an amount of 1/100 per volumewas added from day 2 from the start of the floating culture. Theaggregate was transferred to a serum-free medium without containing asubstance inhibiting the Wnt signal pathway on day 12 from the start ofthe floating culture, fetal calf serum in an amount of 1/10 per volumewas added and cultured. The floating culture was performed in a mediumcontaining a substance acting on the Wnt signal pathway (3 μM CHIR99021)and a substance acting on the Activin signal pathway (RecombinantHuman/Mouse/Rat Activin A (R&D systems #338-AC) 100 ng/ml) from day 15.

(Results)

When produced by the above-mentioned method, retinal pigment epitheliataking on black color emerged on surfaces of almost all the aggregates.Furthermore, almost all the surfaces of the aggregates were covered withretinal pigment epithelia (FIG. 11), and retinal pigment epithelia wereproduced at surprisingly high efficiency.

Example 12 Transplantation into Eye of Retinal Pigment EpitheliumProduced from Human ES Cell

After incision of the sclera of an eyeball, an injection needle wasinserted from the sclera incision into the vitreous to lower theintraocular pressure. An intraocular perfusion fluid was injected fromthe sclera incision into subretinal space with a cell transplantationneedle to artificially form a shallow retinal detachment state. Theretinal pigment epithelium is transplanted with a cell transplantationneedle or a cell sheet transplantation device into the space formed.

INDUSTRIAL APPLICABILITY

According to the present invention, a retinal tissue, optic-cup-likestructure, retinal layer-specific neural cell or retinal pigmentepithelium can be produced at high efficiency. The production method ofthe present invention is highly useful since it efficiently produces acell group (such as photoreceptor and optic nerve) constituting aretinal tissue, for the purpose of toxicity or drug efficacy evaluationof a chemical substance, etc., a cell treatment and so on, as well asefficiently produces a retinal tissue to be a “tissue material” to beused for tests and treatments for the purpose of application to atoxicity or drug efficacy evaluation using a retinal tissue with atissue structure, and to a transplantation material for a retinal tissuetransplantation treatment.

This application is based on patent application Nos. 2011-258209,2011-258210, 2011-258211, 2011-258212, 2012-043080, 2012-043081,2012-043082 and 2012-043083 filed in Japan, the contents of which areincorporated in full herein.

1. A method for producing a retinal tissue, comprising the followingsteps (1) to (3): (1) a first step of subjecting pluripotent stem cellsto floating culture in a serum-free medium containing a substanceinhibiting the Wnt signal pathway to form an aggregate of pluripotentstem cells, (2) a second step of subjecting the aggregate formed in thefirst step to floating culture in a serum-free medium containing abasement membrane preparation, and (3) a third step of subjecting theaggregate cultured in the second step to floating culture in aserum-containing medium.
 2. A method for producing an optic-cup-likestructure, comprising a step of subjecting the retinal tissue obtainedby the method according to claim 1 to floating culture in a serum-freemedium or serum-containing medium each containing a substance acting onthe Sonic hedgehog signal pathway and a substance acting on the Wntsignal pathway.
 3. A method for producing a retinal pigment epithelium,comprising a step of subjecting the retinal tissue obtained by themethod according to claim 1 to floating culture in a serum-free mediumor serum-containing medium each containing a substance acting on the Wntsignal pathway (wherein said serum-free medium and serum-containingmedium are free of a substance acting on the Sonic hedgehog signalpathway).
 4. The method according to claim 3, wherein said serum-freemedium or serum-containing medium containing a substance acting on theWnt signal pathway further comprises a substance acting on the Activinsignal pathway.
 5. The method according to claim 1, wherein saidpluripotent stem cells are primate pluripotent stem cells.
 6. The methodaccording to claim 1, wherein said pluripotent stem cells are humanpluripotent stem cells.
 7. The method according to claim 1, wherein saidbasement membrane preparation is at least one extracellular matrixmolecule selected from the group consisting of laminin, type IVcollagen, heparan sulfate proteoglycan and entactin.
 8. The methodaccording to claim 1, wherein said first step to the third step areperformed in the presence of Knockout serum replacement.
 9. A method forproducing a retinal layer-specific neural cell, comprising bringing aretinal progenitor cell contained in a retinal tissue derived from aprimate pluripotent stem cell into contact with a substance inhibitingthe Notch signal pathway.
 10. The method according to claim 9, whereinsaid substance inhibiting the Notch signal pathway is a gamma secretaseactivity inhibitory substance.
 11. The method according to claim 9,wherein said substance inhibiting the Notch signal pathway isN—[N-(3,5-difluorophenacetyl)-L-alanyl]-5-phenylglycine t-butyl ester.12. The method according to claim 9, wherein said primate is human. 13.The method according to claim 9, wherein the retinal layer-specificneural cell is a photoreceptor.
 14. The method according to claim 9,wherein the retinal layer-specific neural cell is a ganglion cell. 15.The method according to claim 9, wherein the retinal progenitor cellcontained in the retinal tissue derived from a primate pluripotent stemcell is a retinal progenitor cell contained in a retinal tissue producedby the following steps: (1) a first step of subjecting primatepluripotent stem cells to floating culture in a serum-free mediumcontaining a substance inhibiting the Wnt signal pathway to form anaggregate of primate pluripotent stem cells, (2) a second step ofsubjecting the aggregate formed in the first step to floating culture ina serum-free medium containing a basement membrane preparation, (3) athird step of subjecting the aggregate cultured in the second step tofloating culture in a serum-containing medium, (4) a fourth step ofsubjecting the aggregate cultured in the third step to floating culturein a serum-free medium or serum-containing medium each containing asubstance acting on the Sonic hedgehog signal pathway and a substanceacting on the Wnt signal pathway, to form an optic-cup-like structure,and (5) a step of subjecting the optic-cup-like structure formed in thefourth step to floating culture.
 16. A reagent for evaluating toxicityor drug efficacy, comprising a retinal tissue, an optic-cup-likestructure, a retinal pigment epithelium or a retinal layer-specificneural cell produced by the method according to claim
 1. 17. A methodfor evaluating toxicity or drug efficacy of a test substance, comprisingbringing a retinal tissue produced by the method according to claim 1into contact with the test substance, and examining the influence of thesubstance on the tissue, structure or cell.
 18. A therapeutic agent fora disease due to a disorder of a retinal tissue, comprising a retinaltissue produced by the method according to claim
 1. 19. A method fortreating a disease due to a disorder of a retinal tissue, comprisingtransplanting an effective amount of a retinal tissue produced by themethod according to claim 1 to a target in need of the transplantation.20. A retinal tissue produced by the method according to claim 1.